Temozolomide (TMZ) combined with radiation therapy is the standard of care for newly diagnosed patients with glioblastoma multiforme (GBM). A key cytotoxic lesion induced by TMZ is repaired by the O6- methylguanine-methyltransferase (MGMT) DNA repair protein, and disruption of MGMT activity significantly increases the sensitivity of tumors to TMZ. Thus, understanding the regulatory mechanisms controlling MGMT activity is of critical clinical importance. An aspect of this regulation that is of fundamental significance involves MGMT methylation, and several studies have demonstrated an association between MGMT promoter methylation and improved GBM patient survival. However, the methylation specific PCR (MS-PCR) reaction that is used most often to interpret MGMT promoter methylation status examines only 9 of 98 potential CpG methylation sites. Because changes in MGMT promoter methylation outside of the region evaluated by MS-PCR can dramatically affect MGMT expression, we hypothesize that a comprehensive evaluation of CpG island methylation will provide a more robust predictor of TMZ response. To further study the influence of MGMT methylation on TMZ sensitivity, the survival benefit of TMZ therapy has been evaluated in an intracranial xenograft therapy model using a panel of 17 human GBMs that were established and maintained by serial passage in the flank of nude mice. Our preliminary data suggest that methylation at 4 MGMT promoter CpG sites, which are not queried by the routinely-used MGMT MS-PCR analysis, is a more accurate predictor of individual tumor TMZ responsiveness. The primary objective of the studies associated with this application is to conclusively address the relationship between GBM response to TMZ and MGMT methylation by identifying the CpG methylation sites that are critical to regulating MGMT expression. For this purpose, the following experimental plan is put forward. In aim 1, key CpG methylation sites predictive of survival will be identified using GBM tumor specimens from patients treated with TMZ + radiation on 3 prospective clinical trials. In aim 2, the influence of MGMT methylation patterns on TMZ sensitivity and MGMT mRNA and protein expression will be evaluated our panel of GBM xenografts that collectively recapitulate the range of TMZ responses observed in patient clinical trials. Aim 3 studies will focus on the use of xenografts for investigating relationships between changes in MGMT CpG methylation patterns and acquired resistance to therapy, which has been observed in association with our preliminary xenograft investigations. In total, this study plan will provide a definitive delineation of MGMT CpG methylation patterns that consistently and reliably predict tumor response to TMZ therapy, and will address the role of changes to MGMT CpG methylation in acquired resistance to TMZ. The addition of temozolomide to standard radiation therapy significantly prolongs survival in a subset of patients with glioblastoma multiforme, which is the most lethal form of brain cancer. The studies planned are designed to develop a robust predictor of temozolomide responsiveness in patients that could be used to customize therapy for patients with this disease. Investigations into the mechanism of temozolomide drug resistance also will provide the foundation for future development of novel temozolomide sensitizing strategies to further improve the cure rate for patients with glioblastoma multiforme.